Binding and spreading by WT and βTD mutant CD11b/CD18 to iC3b-coated plates. (A) Histograms showing the relative binding of mock, WT, and mutant CD11b/CD18-expressing cells to iC3b-coated plates in buffer containing 1 mM each of Ca²⁺ and Mg²⁺ or 1 mM Mn²⁺ and expressed as the absolute number of adherent cells per well. Each histogram represents mean ± SD of triplicate determinations from a representative experiment (1 of 2 performed). The methods are detailed in “Materials and methods.” No iC3b binding was seen with mock, WT, or mutant CD11b/CD18 in the presence of EDTA (not shown). (B) Histograms showing the relative level of spreading by mock, WT, and mutant CD11b/CD18 cells to iC3b-coated plates in buffer containing 1 mM each of Ca²⁺ and Mg²⁺ and expressed as a percent of spread cells in the total field (more than 200 cells examined in multiple fields using phase-contrast microscopy). Spreading results are reported as histograms representing mean ± SD of triplicate experiments. The methods are detailed in “Materials and methods.” (C) Phase-contrast microscopy images (visualized using a Nikon Eclipse E800 microscope [Nikon, Melville, NY] equipped with a 20×/0.50 numerical aperture objective in TBS) showing the morphology of the cells (mock, WT, and mutant CD11b/CD18) after adhering to iC3b-coated plates for about 1.5 hours in buffer containing 1 mM each of Ca²⁺ and Mg²⁺. Images were acquired using SPOT version 4.5 (Diagnostic Instruments, Sterling Heights, MI) and were cropped using Adobe Photoshop version 5.0 (Adobe Systems, San Jose, CA).